Synthesis of C6-modified mannose 1-phosphates and evaluation of derived sugar nucleotides against GDP-mannose dehydrogenase

Sufferers of cystic fibrosis are at significant risk of contracting chronic bacterial lung infections. The dominant pathogen in these cases is mucoid Pseudomonas aeruginosa. Such infections are characterised by overproduction of the exopolysaccharide alginate. We present herein the design and chemoenzymatic synthesis of sugar nucleotide tools to probe a critical enzyme within alginate biosynthesis, GDP-mannose dehydrogenase (GMD). We first synthesise C6-modified glycosyl 1-phosphates, incorporating 6-amino, 6-chloro and 6-sulfhydryl groups, followed by their evaluation as substrates for enzymatic pyrophosphorylative coupling. The development of this methodology enables access to GDP 6-chloro-6-deoxy-ᴅ-mannose and its evaluation against GMD.


S1. General experimental
All reagents and solvents which were available commercially were purchased from Acros, Alfa Aesar, Fisher Scientific, Sigma Aldrich or TCI. All reactions in non-aqueous solvents were conducted using oven-dried glassware with a magnetic stirring device under an inert atmosphere of nitrogen passed through a drying column using a vacuum manifold. Solvents were purified by passing through activated alumina columns and used directly from a Pure Solv-MD solvent purification system and were transferred under nitrogen unless otherwise stated. Reactions were followed by thin layer chromatography (TLC) using Merck silica gel 60 F 254 analytical plates (aluminium support) and were developed using short wave UV radiation (245 nm) and/or 10% sulfuric acid in methanol/∆. Purification via flash column chromatography was conducted manually using Sigma Aldrich silica gel 60 (0.040-0.063 mm) under a positive pressure of compressed air or via automation using a Büchi Reveleris X2 or a Büchi Pure C-815 Flash with pre-packed silica cartridges. Purification via strong ion exchange (SAX) chromatography was conducted using a Thermo Scientific™ HyperSep™ SAX 500 mg cartridge (column volume = 5 mL) with deionized water followed by aqueous NH 4 HCO 3 (1.0 M). Purification via reversed phase separation was conducted using a Thermo Scientific™ HyperSep™ C18 cartridge (column volume = 5 mL) with deionized water followed by EtOAc and MeCN. Optical activities were recorded on an automatic Rudolph Autopol I or Bellingham and Stanley ADP430 polarimeter (concentration in g/100mL). 1 H NMR spectra were recorded at 400 MHz, 13 C NMR spectra at 100 MHz, and 31 P NMR spectra at 161 MHz respectively using Bruker Magnet system 400'54 Ascend. 1 H NMR resonances were assigned with the aid of S2 gDQCOSY. 13 C NMR resonances were assigned with the aid of gHSQCAD. Coupling constants are reported in hertz. Chemical shifts (δ, in ppm) are standardized against the deuterated solvent peak. NMR data were analyzed using Mestrenova. 1 H NMR splitting patterns were assigned as follows: br. s (broad singlet), s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), app. t (apparent triplet), t (triplet), quartet (q) or m (multiplet and/or multiple resonances). HRMS (ESI) were obtained on Agilent 6530 Q-TOF, LQT Orbitrap XL1 or Waters (Xevo, G2-XS TOF or G2-S ASAP) Micromass LCT spectrometers using a methanol mobile phase in positive/negative ionization modes, as appropriate.
Proteins were eluted with 20 mM HEPES (pH 7.5) and 150 mM NaCl at the flow rate of 1 mL/min. GMD containing fractions were combined and concentrated to ≈4.5 mg/mL (concentration determined by Pierce™ BCA assay, ThermoFisher or Bradfords Assay, Sigma).
Concentrated GMD was then divided into aliquots and stored at −80°C until required in 10% glycerol.

GMD inhibition assay Assay protocol
The assay was performed in 96-well flat bottomed, non-binding, polystyrene microtiter plates  GMD alkylation by iodoacetamide Figure S4: Deconvoluted protein LC-MS of GMD following overnight incubation with iodoacetamide (10 equiv) showing multiple surface-exposed alkylation sites. S12

S5. X-Ray crystallography data
Crystal and refinement parameters are given in Table S1. All data were collected on a Bruker D8 Quest ECO diffractometer using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) and a Photon II-C14 CPAD detector. Crystals were mounted on Mitegen micromounts in NVH immersion oil, and all collections were carried out at 150 K using an Oxford cryostream. Data collections were carried out using φ and ω scans, with collections and data reductions carried out in the Bruker APEX-3 suite of programs. 5 Multi-scan absorption corrections were applied for all datasets using SADABS unless otherwise stated. 6 The data were solved with the intrinsic phasing routine in SHELXT, 7 and all data were refined on F 2 with full-matrix least squares procedures in SHELXL, 8 operating within the OLEX-2 GUI. 9 All non-hydrogen atoms were refined with anisotropic displacement parameters. Carbonbound hydrogen atoms were placed in riding positions and refined with isotropic displacement parameters equal to 1.2 or 1.5 times the isotropic equivalent of their carrier atom. Crystals of 16 exhibited unavoidable non-merohedral twinning related by a 180 degree rotation which could not be mechanically separated. The two domains were indexed and their contributions to each reflection were separated using TWINABS, 10